Interactome of NOP58 WT vs KA_AR mutant
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Franck Vandermoere
LAB HEAD: Edouard Bertrand
PROVIDER: PXD019415 | Pride | 2020-12-14
REPOSITORIES: Pride
Similar Datasets
Project description:The PAQosome is composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and the RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 binds several RUVBL1/2 mutants except those unable to hydrolyze ATP. Moreover, while it robustly binds both yeast and human NOP58, it makes only weak interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Transient expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that select NOP58 to promote box C/D snoRNP assembly.
2020-12-14 | PXD019389 | Pride
Project description:The PAQosome is composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and the RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 binds several RUVBL1/2 mutants except those unable to hydrolyze ATP. Moreover, while it robustly binds both yeast and human NOP58, it makes only weak interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Transient expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that select NOP58 to promote box C/D snoRNP assembly.
2020-12-14 | PXD019416 | Pride
Project description:The PAQosome is composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and the RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 binds several RUVBL1/2 mutants except those unable to hydrolyze ATP. Moreover, while it robustly binds both yeast and human NOP58, it makes only weak interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Transient expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that select NOP58 to promote box C/D snoRNP assembly.
2020-12-14 | PXD019414 | Pride
Project description:The PAQosome is composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and the RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 binds several RUVBL1/2 mutants except those unable to hydrolyze ATP. Moreover, while it robustly binds both yeast and human NOP58, it makes only weak interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Transient expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that select NOP58 to promote box C/D snoRNP assembly.
2020-12-14 | PXD019390 | Pride
Project description:The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RuvBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.
2020-12-11 | GSE161155 | GEO
Project description:NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis
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Project description:RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression prognostic of poor survival. Utilizing a highly specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of their ATPase activity impedes RPA loading at replication forks, decreases fork stability, and ultimately results in replication catastrophe and cancer cell death. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.
2019-12-18 | GSE107637 | GEO
Project description:The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: Dyskerin, NHP2, NOP10 and GAR1. The assembly of the H/ACA RNPs is a stepwise process which requires several assembly factors: SHQ1, NAF1, the Survival of Motor Neuron complex SMN, and the R2TP complex. It implicates the co-transcriptional assembly of a pre-particle containing the nascent RNAs, Dyskerin, NOP10, NHP2 and NAF1. The latest keeps the H/ACA RNP inactive, and needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the Cajal bodies. In this study, we explore in details the molecular mechanism leading to the assembly of mature box H/ACA RNPs. We performed extensive analysis of GAR1, NHP21 and NAF interactomes by quantitative stable isotope labeling by amino acids in cell culture proteomic analyses (SILAC), and revealed new assembly intermediates: 1) an early protein-only pre-H/ACA RNP complex containing the core proteins DKC1, NOP10 and NHP2, and the assembly factors SHQ1 and NAF1, and 2) a late assembly intermediates containing the RNA, the core proteins and NAF1. We showed that the SMN complex is associated with late forming H/ACA pre-RNP complexes containing GAR1. Moreover, our study identifies new proteins highly and specifically associated with GAR1, NHP2 and NAF1, reflecting their importance in box H/ACA assembly or function, such as the PRMT1 and PRMT5 arginine methylases. MS analysis of purified GAR1 revealed new sites of arginine methylations in the two GAR domains of GAR1, and showed that most of these methylated arginines exist both in mono-methylated and di-methylated forms in cells. By different methods, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs. Therefore, GAR1 methylation should be important for its function.
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Project description:The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: Dyskerin, NHP2, NOP10 and GAR1. The assembly of the H/ACA RNPs is a stepwise process which requires several assembly factors: SHQ1, NAF1, the Survival of Motor Neuron complex SMN, and the R2TP complex. It implicates the co-transcriptional assembly of a pre-particle containing the nascent RNAs, Dyskerin, NOP10, NHP2 and NAF1. The latest keeps the H/ACA RNP inactive, and needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the Cajal bodies. In this study, we explore in details the molecular mechanism leading to the assembly of mature box H/ACA RNPs. MS analysis of purified GAR1 revealed new sites of arginine methylations in the two GAR domains of GAR1, and showed that most of these methylated arginines exist both in mono-methylated and di-methylated forms in cells. By different methods, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs. We performed extensive analysis of GAR1, NHP2 and NAF1 proteomes by quantitative stable isotope labeling by amino acids in cell culture proteomic analyses (SILAC), and revealed new assembly intermediates: 1) an early protein-only pre-H/ACA RNP complex containing the core proteins DKC1, NOP10 and NHP2, and the assembly factors SHQ1 and NAF1, and 2) a late assembly intermediates containing the RNA, the core proteins and NAF1. We showed that the SMN complex is associated with late forming H/ACA pre-RNP complexes containing GAR1. Moreover, our study identifies new proteins highly and specifically associated with GAR1, NHP2 and NAF1, suggesting their importance in box H/ACA assembly or function, such as the PRMT1 and PRMT5 arginine methylases.
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Project description:The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: Dyskerin, NHP2, NOP10 and GAR1. The assembly of the H/ACA RNPs is a stepwise process which requires several assembly factors: SHQ1, NAF1, the Survival of Motor Neuron complex SMN, and the R2TP complex. It implicates the co-transcriptional assembly of a pre-particle containing the nascent RNAs, Dyskerin, NOP10, NHP2 and NAF1. The latest keeps the H/ACA RNP inactive, and needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the Cajal bodies. In this study, we explore in details the molecular mechanism leading to the assembly of mature box H/ACA RNPs. MS analysis of purified GAR1 revealed new sites of arginine methylations in the two GAR domains of GAR1, and showed that most of these methylated arginines exist both in mono-methylated and di-methylated forms in cells. By different methods, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs. We performed extensive analysis of GAR1, NHP2 and NAF1 proteomes by quantitative stable isotope labeling by amino acids in cell culture proteomic analyses (SILAC), and revealed new assembly intermediates: 1) an early protein-only pre-H/ACA RNP complex containing the core proteins DKC1, NOP10 and NHP2, and the assembly factors SHQ1 and NAF1, and 2) a late assembly intermediates containing the RNA, the core proteins and NAF1. We showed that the SMN complex is associated with late forming H/ACA pre-RNP complexes containing GAR1. Moreover, our study identifies new proteins highly and specifically associated with GAR1, NHP2 and NAF1, suggesting their importance in box H/ACA assembly or function, such as the PRMT1 and PRMT5 arginine methylases.
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